The present invention relates to a method for attaching biomolecules to hydrophobic surfaces using a fusion protein which encodes for lipid attachment via post-translational modification.
Many routine medical immunoassays are performed on polystyrene or latex surfaces, most commonly involving antibodies immobilized on a carrier such as plastic beads or containers. Conventionally, simple adsorption is the most common method for attaching antibodies to a hydrophobic surface such as a polystyrene container or latex beads. Adsorption is very simple, but it is not always efficient. High concentrations of the protein to be adsorbed are often necessary, and proteins often denature and lose activity when they are adsorbed to a hydrophobic surface. If proteins are adsorbed at a high density, desorption is also a problem, especially if the proteins are exposed to solutions containing detergents.
To overcome the problem with desorption, it is currently necessary to form a covalent attachment of the molecules to the surface. These procedures require a surface with suitable reactive groups available. These groups can be introduced to materials such as polystyrene by very harsh chemical treatment or they can be introduced during the initial manufacture of the material to be coated. Even if the reactive groups exist on the surface, a relatively complex chemistry requiring a heterobifunctional crosslinking reagent is required. This type of crosslinking is generally not orientationally specific, so that often the amount of active material bound to the surface is reduced by 50%.
Huang et al., in J. Biol. Chem 255: 8015-8018 (1980), describe coupling fatty acyl groups in lipids to appropriately exposed sulfhydryl and amino acid groups in a protein molecule with a bifunctional reagent. However, in such chemical coupling procedures, the conjugate often forms a heterogeneous complex in terms of number and location of lipid moieties. As a result, this treatment may lead to a loss or decrease in antigen-binding properties.
Other methods have been tried to attach biomolecules such as proteins to substrates, many of which involve using a modified protein as a coating material. For example, Piersbacher et al., in U.S. Pat. No. 4,589,881, disclose polypeptides which have the cell-attachment-promoting activity of fibronectin. These polypeptides are used to prepare a substrate to which cells will attach. In this case the polypeptide is the coating to which biomolecules can be attached.
In another method, Fischer, in U.S. Pat. No. 4,264,766, discloses immunological reagents comprising discrete particles of a latex carrier to which a water-soluble polyhydroxy compound is covalently bound, having condensed thereto a known immunologically active material.
Furcht et al., in U.S. Pat. No. 5,294,551, disclose polypeptides which can bind heparin and promote cellular adhesion and neurite outgrowth. Substrates are coated with these polypeptides in order to culture or grow cells.
Means et al., in U.S. Pat. No. 4,808,530, disclose immobilizing biologically active proteins by reacting the protein with an imidoester or imidothioester having a hydrophobic moiety to form a hydrophobic amidine derivative of the protein, and then adsorbing the protein derivative to a hydrophobic surface.
Anumula et al., in U.S. Pat. No. 5,811,246, disclose a process for immobilizing a compound onto the surface of ELISA plates. The compound is in the form of a compound carrier complex with either avidin-biotin or streptavidin-biotin.
Mootadian et al., in U.S. Pat. No. 5,853,744, disclose a method of covalently attaching a biomolecule to a support by covalently attaching a biomolecule to a support material, attaching a photoreactive crosslinking agent to the immobilized biomolecule to form an immobilized photoreactive analog of the biomolecule, removing the photoreactive analog of the biomolecule from the support material, and attaching the photoreactive analog of the biomolecule to a substrate surface.
Goetnick et al., in U.S. Pat. No. 5,872,094, disclose attaching cartilaginous tissue or protein to a surface by administering a polypeptide capable of promoting the binding of a complex of a proteoglycan and hyaluronic acid to collagen. The administered polypeptide is a fusion polypeptide containing a fragment of cartilage matrix protein and a fragment of link protein. In this case the protein is administered to a patient to repair diseased or injured tissue by promoting the binding of a complex of proteoglycan and hyaluronic acid to collagen.
Takkinen et al., in Protein Engineering 4:837-841 (1991) disclose preparing an active single-chain antibody containing a cellulase linker domain which is secreted by E. coli. These molecules were designed to allow incorporation of the single chain antibodies into lipid micelles. The lipophilic antibody was genetically engineered and found to be active and capable of functioning in lipid micelles. However, this protein did not provide an enhanced signal over unbound antibody.
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art.
It is another object of the present invention to provide a simple method for attaching biomolecules to hydrophobic surfaces.
It is a further object of the present invention to provide a simple method for attaching biomolecules to hydrophobic surfaces using a fusion protein which encodes for lipid attachment via post-translational modification.
The present invention involves a recombinant protein that is a fusion protein that contains a bioactive domain such as an immunoglobulin binding domain and a domain that is post-translationally modified to become lipid-tagged. This creates a molecule that has both a domain with highly specific binding capabilities and a domain which is very hydrophobic in nature. Molecules of this type previously were thought to be useful only for preparing lipid micelles or membranes. However, the present inventors have discovered that this type of molecule can be used to coat hydrophobic surfaces such as polystyrene easily and effectively.
The advantage of surface modification by this method is its simplicity. Once the lipid protein is added to the component to be coated, nearly instantaneously the desired protein is adsorbed to the surface.